Metabolic engineering permits production of compounds through manipulation of biochemical reactions (e.g., biosynthetic pathways) in a cell. Nonetheless, production of certain compounds may conflict with essential cellular goals. For example, diversion of nutrients and energy for the production of a compound may result in a shortage of those substrates and cofactors for production of biomass. The engineered organism may either evolve away from producing the compound of interest or grow sub-optimally. To address this issue, cell-free systems have been developed for the in vitro production of compounds through coordinated expression of proteins in a biosynthetic pathway. One caveat to both in vivo and in vitro bioproduction systems is that many key proteins that divert flux from a biosynthetic pathway are also important or even essential for cell growth. Deletion or inactivation of these proteins is often difficult or impossible because doing so results in reduced cell growth or viability. One way to inactivate proteins is through protease-mediated inactivation. Protease-mediated inactivation of a target protein can be achieved through the incorporation of a protease recognition site in the primary amino acid sequence of the target protein. The protease recognition site can be incorporated into the primary sequence such that the resulting protein is active in the absence of a protease that cleaves the recognition site and inactive in the presence of the protease. Such engineered or recombinant target proteins are particularly useful for the cell-free synthesis of compounds of interest.